Industrially Scaleable Process for the Purification and Refolding of Inclusion Body Recombinant Prot

1
Industrially Scaleable Process for the
Purification and Refolding of Inclusion Body
Recombinant Protein BELINDA C. CLARKE1, GARETH M.
FORDE1, CHARLES HARDY2, ALEC DREW2, ROBYN
OHEHIR2 1 Department of Chemical Engineering,
Melbourne, Victoria, 3800, Australia. 2 CRC for
Asthma, Dept. of Immunology, Central and Eastern
Clinical School, Monash University,


The Problem There is a large and increasing
demand for the production of recombinant protein
for use in pre-clinical, clinical and commercial
applications. Traditionally, inclusion body
recombinant protein is manufactured using
complicated laboratory scale unit operations.
However, the biotechnology industry is maturing
and patent protection periods are beginning to
expire. This is driving a global shift towards
higher productivity, commercially viable
processes.

The Objective The aim of this research is to
develop, optimise and analyse a scaleable,
commercially viable process for the production of
recombinant protein from inclusion bodies.
Initially, this process will be used to produce a
latex allergen (Hev b 6) for pre-clinical studies
at the CRC for Asthma. The protein needs to be of
high purity and concentration (i.e. no
endotoxins), biologically active, and in a
refolded formed.

What is an inclusion body? When E.coli is
transformed to manufacture large amounts of
recombinant protein, the protein sometimes forms
dense aggregates of insoluble misfolded proteins,
known as inclusion bodies. The benefit from a
production aspect of inclusion bodies is that
they allow high protein concentrations, protect
sensitive proteins from proteolytic (enzymatic)
degradation and protect the cell from any toxic
proteins. However, the challenge is to solubilise
and refold this protein into its correct active
form.
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6. Refolding Vessel The protein is renatured by
decreasing the concentration of the denaturant
urea. This step will be performed on the
adsorption column to help prevent aggregation.
1. Fed Batch Fermentation Cell lines transformed
with the appropriate DNA supplied by the CRC for
Asthma are grown and induced to express Hev b 6.
The Process

2. Chemical Disruption and Extraction The cells
are lysed to release the inclusion bodies and the
protein is denatured using a chemical wash
containing urea. Spermine is also added to reduce
the negative effects of DNA on the adsorption
column efficiency.


5. Expanded Bed Adsorption Column The protein
is purified using an automated chromatography
unit (Biorad DuoflowTM) via an affinity agarose
adsorbent (Ni charged resin) which has
specificity for the His-tagged Hev b 6 protein.

3. Conditioning CaCl2 is added to remove the
EDTA, as EDTA decreases the efficiency of the
adsorption column.

4. Filtration Removes precipitates and the
DNA-spermine complexes. Some concentration of the
feedstock will also occur.


Protein Refolding The most significant challenge
of this process is the refolding step as ? Small
amounts of contaminants can decrease the yield of
refolded protein (for example, contaminants can
cause aggregation and proteases cause degradation
of the protein). ? The protein must be in an
environment that favours the formation of the
correctly folded proteins over the misfolded.
This may require chaperones, metal ions etc. ?
Refolding conditions vary with proteins.
Preliminary Results Chromatographic purification
(2 ml packed bed) of the Hev b 6 protein has been
performed as an analytical method to facilitate
optimisation studies of protein expression and to
bench-mark against the original CRC for Asthma
protocol. These results will determine optimal
operating parameters (e.g. expression time,
induction cell density, inducer concentration)
for the scaleable protein production process.
Chromatograph for Purification of Hev b 6-His
Tagged Protein
Indication of protein concentration leaving column
Fractions collected and analysed using Bradford
Assay and SDS-Page to identify the products
Bound protein eluted (total of approximately 3.8
mg protein from Bradford Assay)
Acknowledgement